N-Terminal Phosphorylation of the Dopamine Transporter Is Required for Amphetamine-Induced Efflux

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake

Dopamine transporter regulation by amphetamine: Phosphorylation and trafficking events.

The dopamine transporter (DAT), the site of action of the psychostimulant drug amphetamine (AMPH), undergoes highly dynamic and complex regulation. There is extensive investigation of the compensatory effects of persistent amphetamine administration and protein kinase C on the down-regulation of the influx function of DAT. However, we find that amphetamine and protein kinase C have rapid and robust effects on the efflux function of DAT. This thesis describes the effects of amphetamine and protein kinase C on rapid DAT trafficking and DAT-mediated efflux of dopamine. In the first study, using classical pharmacological inhibitors, I determined that classical PKC isozymes are essential for AMPH-stimulated DA efflux. A highly selective inhibitor permitted identification of the important isoform as being PKCbeta. Using hDAT HEK cells that transiently overexpress either PKCalpha, PKCbetaI, or PKCbetaII, it was determined that overexpression of PKCbetaII potentiated AMPH-stimulated DA efflux. In the second study, the AMPH-elicited rapid effects (3H]DA uptake. The AMPH-stimulated increase in cell surface DAT appears to be dependent on PKCbeta activity. In the third study, using a mutant DAT in which the first 22 N-terminal amino acids were truncated, I determined that deletion of the N-terminal of DAT results in an 80% loss in AMPH-stimulated efflux without impairing [ 3H]DA uptake when compared to wildtype. A collaborative effort with Dr. Jonathan Javitch, Columbia University, demonstrated that phosphorylation of N-terminal serines was crucial for efflux. These results establish there are separate structural determinants for uptake versus efflux functions of DAT and that the regulation is through phosphorylation. In summary, the work in this thesis has delineated the isoform of protein kinase C that regulates AMPH-stimulated DA efflux, proven that there are separate structural determinants in the protein regulating inward and outward efflux and made the original observation that AMPH elicits rapid functional effects on trafficking of DAT. These findings are important for DA- and DAT-related pathologies such as schizophrenia and substance abuse.Ph.D.Health and Environmental SciencesPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125394/2/3192669.pd

N-Terminal Truncation of DAT Reduces AMPH-Induced Currents and DA Efflux

<div><p>Cells were voltage clamped with a whole-cell patch pipette while an amperometric electrode was placed onto the cell membrane. The internal solution of the whole-cell patch pipette contained 2 mM DA.</p> <p>(A) Representative trace of AMPH-induced whole-cell current obtained from FLAG-DAT cells upon AMPH (10 μM) bath application. The membrane potential of the cell was stepped to +100mV from a holding potential of –20 mV.</p> <p>(B) Oxidation current acquired concomitantly to the whole-cell current represented in panel A.</p> <p>(C and D) Representative current traces (whole-cell and amperometric, respectively) obtained from FLAG-del22-DAT cells using the same experimental protocol as in (A) and (B).</p></div

N-Terminal Truncation of DAT Impairs AMPH-Induced DA Efflux

<p>Cells were preloaded with 15 μM DA and superfused with AMPH at concentrations ranging from 1 to 100 μM. AMPH-induced DA efflux was defined as the amount of DA released in response to the given concentration of AMPH minus the baseline value. Baseline DA release did not differ between FLAG-HA-DAT and FLAG-DAT (13.2 ± 2.9 and 10.2 ± 1.8, respectively; <i>n =</i> 18). The V_max of efflux was 31.1 ± 4.6 and 128.3 ± 12.0 pmol/mg protein/fraction (F(2,27) = 52.6, <i>p</i> < 0.0001) with a K_m for amphetamine of 7.8 ± 4.1 and 7.6 ± 2.2 μM, for FLAG-HA-DAT and FLAG-DAT, respectively (<i>n</i> = 4). For [³H]DA uptake, the V_max was 15.4 ± 2.5 and 18.3 ± 2.2 pmol/min/mg protein with a K_m of 1.2 ± 0.8 and 1.1 ± 0.4 μM for FLAG-HA-DAT and FLAG-DAT, respectively (F(2,49) = 1.78, <i>p</i> > 0.17).</p